Detection Of Anti-drug Antibodies Research Articles

DOP029 Clinical relevance of detecting anti-infliximab antibodies with a drug-tolerant assay: post-hoc analysis of the taxit trial

Background: Anti-drug antibodies (ADA) may develop in up to 51% of patients treated with infliximab maintenance therapy and are associated with infusion reactions and impaired response. Drug-sensitive assays do not detect ADA in the presence of drug and underestimate ADA formation. Drug-tolerant assays have therefore been developed and markedly increased the detection of ADA, although their clinical relevance remains to be shown. Our goal was to evaluate the clinical relevance of anti-drug antibodies (ADA) measured using a drug-tolerant assay in comparison to a drug-sensitive assay in a post-hoc analysis of the Trough Concentration Adapted Infliximab Treatment (TAXIT) randomized controlled trial. Methods: Patients who presented with an infliximab trough concentration (TC) <3 μg/mL at screening (n=76) underwent dose escalation to achieve therapeutic TCs between 3–7 μg/mL prior to randomization. Serum samples were re-analyzed at screening, after optimization and one year after randomization using a drug-tolerant ADA assay. Results: Using a drug-tolerant assay, the immunogenicity detection rate increased from 21% (drug-sensitive assay) to 63% at screening, from 0% to 51% after optimization and from 3% to 42% one year after randomization. ADA concentration (median [interquartile range, IQR]) in ADA+ patients grouped into quartiles according to ADA concentration at screening, decreased throughout the study: from 220 [116–737] ng/mL eq at screening to 112 [78–180] ng/mL eq after optimization and to 95 [0–166] ng/mL eq at the end of the study. Patients in ADA Q4 required a higher cumulative infliximab dose (2390 [880–2998] mg) to achieve target TCs (Figure 1) compared to ADA negative patients (450 [365–680] mg, p<0.001) and patients in ADA Q1/Q2 (425 [344–863]/483 [398–719], p<0.001). Figure 1. Cumulative infliximab (IFX) dose required to achieve target concentrations between 3–7 μg/mL A) for ADA+ and ADA- patients, B) across ADA+ quartiles (Q1–Q4) and C) according to the assay (DT, drug-tolerant assay; DS, drug-sensitive assay). However, all but one patient belonging to ADA Q4 were also ADA positive using a drug-sensitive assay. Once dose optimized, patients have similar rates of clinical, biological and endoscopic remission after one year of treatment, regardless of ADA status in a drug-tolerant assay. Conclusions: Upon dose intensification, low concentration ADA, not detectable using a drug-sensitive assay, disappear in more than half of the patients over time and are clinically non-relevant. In contrast, high concentration ADA which are typically also detected in a drug-sensitive assay, persist over time and necessitate a higher cumulative dose and drug cost. In the latter group, proactive drug switching may be more cost-efficient.

Biologic therapies in rheumatic diseases: drug and anti-drug antibody levels and clinical efficacy

Abstract Background: The aim of this study was to evaluate the relevance of drug and anti-drug antibody detection in the clinical management of patients with rheumatoid arthritis (RA) and spondyloarthropaties (SpA) in

Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.

Mitigation of Pre-existing Antibodies to a Biotherapeutic in Non-clinical Species When Establishing Anti-drug Antibody Assay Cutpoint.

Biotherapeutics are known for their potential to induce drug specific immune responses, which are commonly evaluated by the detection of anti-drug antibodies (ADAs). For some biotherapeutics, pre-existing ADAs against drug have been observed in drug-naïve matrix. The presence of pre-existing drug specific antibodies may significantly complicate assessment of the screening ADA assay cutpoint value, which is usually established based on the statistical analysis of signal distribution from the drug-naïve individuals. A Gaussian mixture model-based approach is presented herein to address high prevalence of pre-existing ADAs to a modified monoclonal antibody-based biotherapeutic (m-mAb). A high prevalence of pre-existing anti-m-mAb antibodies was observed in drug-naïve individual cynomolgus monkey serum samples with signal ranging from 100 to 7000 relative light units (RLU, as determined in an electrochemiluminescence readout-based assay). Application of the industry standard statistical algorithm resulted in a relatively high floating screening assay cutpoint factor (CPF) of 9.80, which potentially would have reported a high percent of false negative samples. An alternative, Gaussian mixture model-based approach was applied to identify the least reactive individual samples in the tested population, which resulted in a floating screening assay CPF of 2.35. The low CPF value significantly reduced the risk of reporting false negative results. The proposed Gaussian mixture model-based approach described herein provides an alternate method for the calculation of biologically relevant screening assay CPF when high prevalence of pre-existing drug specific antibodies is observed.

ASSESSING ANTI-TNF TROUGH LEVELS AND ITS APPLICABILITY IN DAILY PRACTICE IN SPONDYLOARTHRITIS PATIENTS

Objective. The purpose of the present study was to assess the relevance of therapeutic drug monitoring in spondyloarthritis patients, by determining drug serum levels and anti-drug antibodies and estimating cut-off values for three TNF inhibitors. Methods. Over one year, we enrolled 100 patients with SpA, under consequent treatment with adalimumab (ADL), etanercept (ETA) or infliximab (IFX). Demographic, clinical (BASDAI, ASDAS) and laboratory (ESR, CRP) data was collected together with drug serum level and anti-drug antibodies using the ELISA technique. The statistical analysis was performed using the SPSS software, version 20.0 with the aid of Student t-test, Spearman and Pearson tests. Results. Out of the study cohort, 35% were on ADL, 33% on IFX, and 32% under ETA treatment. Undetectable drug levels correlated to the presence of anti-drug antibodies and to disease activity scores. There were no identified anti-ETA antibodies. For this study lot trough levels are estimated between 2 and 4 μg/mL for an ASDAS-CRP under 2.1. Conclusion. Serum drug level measurement and anti-drug antibody detection can be used as a completion to a clinician’s tools in assessing disease activity, leading to an optimal and personalized manner of patient management.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Detection of antidrug antibodies against human therapeutic antibodies lacking Fc-effector functions by usage of soluble Fcγ receptor I.

Bridging immunoassays for the detection of antidrug antibodies (ADAs) are limited to detection of bivalent molecules and are prone to interference by drug and soluble targets. Hence, alternative approaches for ADA detection are desired. Materials& methods: A novel ADA assay with secondary Fc detection using human soluble Fcγ receptor I (hsFcγRI) was established and compared with standard bridging assay. Both assays showed consistent results in human and cynomolgus monkey samples. In contrast to the bridging assay, the hsFcγRI-based assay was insensitive to the presence of oligomeric targets and appeared to have better drug tolerance. The hsFcγRI-based ADA assay can serve as alternative screening assay or as orthogonal confirmation method for preclinical and clinical immunogenicity testing of IgG therapeutics lacking Fc effector functions.

Antidrug Antibody Formation in Oncology: Clinical Relevance and Challenges.

Because of the increasing use of biologicals in oncology, many patients are at risk of developing antidrug antibodies (ADAs) during therapy. Although clinical consequences are uncertain, ADAs may affect pharmacokinetics, patient safety, and treatment efficacy. ADA detection and reporting is currently highly inconsistent, which makes it difficult to evaluate the clinical consequences. Standardized reporting of ADA investigations in the context of the aforementioned parameters is critical to understanding the relevance of ADA formation for each drug. Furthermore, the development of trials that specifically aim to investigate clinical prevention strategies in oncology is needed.

PWE-029 Utility of Measurement Anti TNF Drug and Anti Drug Antibody Levels in A Cohort of Patients with Crohn’s Disease: How Does it Affect Clinical Practice?: Abstract PWE-029 Table 1

Introduction It has been recognised that the measurement of anti-TNF drug concentrations and detection of anti drug antibodies may be useful in aiding decision making and therefore optimising clinical outcomes in patients on anti-TNF drugs in whom there is incomplete clinical response or loss of drug therapy. 1 Methods A total of 45 Crohn’s patients attending our tertiary referral IBD clinic had anti TNF drug concentrations and antidrug antibodies levels measured. The indications for testing were incomplete response or loss of response during therapy. We measured drug levels and Anti Drug Antibody levels using the ELISA assay (Immunodiagnostik). Reference ranges for Infliximab >2.0 ug/ml therapeutic and Adalimumab >5.0 ug/ml therapeutic. Total drug antibody levels >10 AU/ml positive. Results We present the patient demographics in Table 1. Drug and ADA levels 44% (n = 20) had therapeutic levels and negative antibodies, 18% (n = 8) had therapeutic levels and positive antibodies, 22% (n = 10) had sub-therapeutic levels and negative antibodies and 16% (n = 7) had sub-therapeutic levels and positive antibodies. Impact of results meaurement of drug and ADA led to change in management in 28/45 (62%) patients. 6 (13%) patients switched therapy,17 (38%) had dose escalation,17 (38%) no treatment change,5 (11%)other outcome. Conclusion Measurement of anti-TNF drug concentrations and antibody status in our patients on Anti TNF resulted in change of management in 28/45 (62%) patients. In the 17 patients with dose escalation in whom outcome was assessed 13 (76%) patients have responded. Measurement of drug concentration and antibody status presents an possible option in the management of patients on anti-TNF who present with loss of response or incomplete response of treatment Reference 1 Vande Casteele N, et al. The relationship between infliximab concentrations, antibodies to infliximab and disease activity in Crohn’s disease. GUT 2015 Oct; 64 (10):1539–45. Disclosure of Interest S. Laird: None Declared, L. Caulfield: None Declared, L. Smith Conflict with: cook, boston and allergan, almirall, J. Winter Conflict with: almirall, reckitt-Benckisaer, ferring, shire, D. Gaya: None Declared, A. Morris Conflict with: Falk,Vifor

Sa1972 Factors Influencing Infliximab and Adalimumab Antibody Formation and 6 Month Retrospective Follow up in a Tertiary IBD Centre

Introduction Infliximab(IFX) and adalimumab(Ada) are established treatments for IBD.However, immunogenicity can lead to anti-drug antibodies(ADAb) which is associated with a poor clinical response.Little is known regarding the factors which influence ADAb formation or the long term outcomes. Methods Patients with detectable ADAb between May 2012 and October 2014 were identified.Assays had been performed using LISA-Tracker DUO Kits which detects free drug levels(DLs) and ADAb only.Patients were analysed for disease phenotype, prior anti-TNF treatment and ADAb formation,and the use of concomitant immunomodulation(IM). Results 330 IFX DLs and ADAb levels from 199 patients and 143 Ada DLs and ADAb levels from 105 patients were analysed.21 positive ADAb, 19 IFX(9.5%) and 2 Ada(1.9%),were detected with a median ADAb of 141.8 U/ml(range 11–200),all DLs were >1.0μg/ml. 47.6% were male, age range 21–84,median 33.85.7% had Crohn’s Disease.In the majority of patients DL and ADAb were measured due to loss of response(66.7%). 6/19 patients who had IFX ADAb had prior exposure to IFX and 1 had prior exposure to Ada.Time to antibody detection ranged from 2–140 weeks(median 14).Of the 6 patients who had previous exposure to IFX,median time to ADAb detection was 6 weeks.Both patients who developed Ada ADAb had prior exposure to Ada,one also had prior exposure to IFX. 18/21 patients were prescribed concomitant IM(2 methotrexate, 16 thiopurines).8/16 on thiopurines had subtherapeutic TGNs( IFX ADAb group(n = 19):11 patients were switched to Ada, all of whom had a clinical response(mean HBI at week 26 = 2.1, no ADAb detected). 1 was switched out of class to vedolizumab and responded well. 3 stopped biologic treatment,1 patient continued on IFX at their own request and 1 patient was dose escalated but failed to respond. 2 patients had concomitant IM optimised,1 of whom on subsequent ADAb testing showed loss of ADAb. Ada ADAb group(n = 2):1 patient stopped anti-TNF due to recurrent infections and the other was lost to follow up. Conclusion 42.9% of patients with ADAb had been exposed to anti-TNF previously,all with a gap of >6 months prior to repeat exposure suggesting this is a risk factor for earlier ADAb formation. All patients switched to Ada from IFX responded well without developing ADAb,highlighting that switching to another anti-TNF is an acceptable option before switching out of class. Our use of concomitant IM is high but 50% of this cohort had subtherapeutic TGNs.The 2 patients in whom a thiopurine was optimised achieved clinical remission while remaining on IFX. However the level at which TGNs should be targeted in this context is still unclear Disclosure of Interest B. Warner: None Declared, E. Johnston: None Declared, J. Digby-Bell: None Declared, Z. Arkir: None Declared, N. Unsworth: None Declared, S. Anderson: None Declared, J. Sanderson: None Declared, P. Irving Grant/research support from: PI has been advisor to, in receipt of educational or research grants from, or invited lecturer for Abbvie, Falk, Ferring, Genetech, Hospira, Merck, Pharmacosmos, Shire, Takeda, Tillotts, Topivert, Vifor and Warner Chilcott, Paid instructor for: as above

Immunogenicity to Biotherapeutics - The Role of Anti-drug Immune Complexes.

Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on prevalence and intensity of established examples of type II and III hypersensitivity reactions that follow the administration of a biotherapeutic. Additionally, we propose methods for the study of immune parameters specific to the biology of ICs that could be of use in conjunction with the detection of ADAs in circulation.

Development of an ELISA-Based Competitive Binding Assay for the Analysis of Drug Concentration and Antidrug Antibody Levels in Patients Receiving Adalimumab or Infliximab

There is increasing interest in measuring both drug and antidrug antibody (ADA) levels in patients receiving anti-tumor necrosis factor treatment as part of algorithms for guiding therapeutic strategies. Many of the current assays for ADA detection have limitations with respect to specificity, sensitivity, and/or laboratory requirements. Specific identification of ADA based on their inhibitory activity in a simple competitive binding assay remains problematic. The development of an enzyme-linked immunosorbent assay (ELISA)-based method for detection of both drug and ADA in patients receiving either adalimumab or infliximab would widen availability of monitoring for these patients. An ELISA for the specific detection of adalimumab and infliximab using widely available reagents was developed. A simple modification for the detection of ADA capable of competitively inhibiting the in vitro binding of drug to solid phase tumor necrosis factor was also developed. Drug and ADA levels were analyzed in patients with rheumatoid arthritis and inflammatory bowel disease. The ELISA specifically detected drug concentrations in patient sera with no evidence of positive or negative interference by rheumatoid factor positive control sera. A subset of those patients with low drug concentrations had detectable levels of ADA with inhibitory activity in a competitive binding assay. Spiking with both drugs confirmed the specificity of the ADA detected. A modified ELISA protocol can be used to for the detection of both drug concentrations and ADA in patients receiving either adalimumab or infliximab. The ELISA incorporates those features identified in the literature as important for the accurate analysis of these antibodies and uses laboratory facilities and reagents that are widely available. It therefore provides a relatively simple and low cost assay for therapeutic drug monitoring of inpatients receiving adalimumab or infliximab.

Utility of a Bayesian Mathematical Model to Predict the Impact of Immunogenicity on Pharmacokinetics of Therapeutic Proteins

The impact of an anti-drug antibody (ADA) response on pharmacokinetic (PK) of a therapeutic protein (TP) requires an in-depth understanding of both PK parameters and ADA characteristics. The ADA and PK bioanalytical assays have technical limitations due to high circulating levels of TP and ADA, respectively, hence, significantly hindering the interpretation of this assessment. The goal of this study was to develop a population-based modeling and simulation approach that can identify a more relevant PK parameter associated with ADA-mediated clearance. The concentration-time data from a single dose PK study using five monoclonal antibodies were modeled using a non-compartmental analysis (NCA), one-compartmental, and two-compartmental Michaelis-Menten kinetic model (MMK). A novel PK parameter termed change in clearance time of the TP (α) derived from the MMK model could predict variations in α much earlier than the time points when ADA could be bioanalytically detectable. The model could also identify subjects that might have been potentially identified as false negative due to interference of TP with ADA detection. While NCA and one-compartment models can estimate loss of exposures, and changes in clearance, the two-compartment model provides this additional ability to predict that loss of exposure by means of α. Modeling data from this study showed that the two-compartment model along with the conventional modeling approaches can help predict the impact of ADA response in the absence of relevant ADA data.

Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies.

Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.

A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays

Biological matrix interference in detection and quantitation immunoassays remains a major challenge in the field of bioanalysis. For example, circulating drug may interfere with the detection of anti-drug antibodies (ADA) and drug target, or ADA may interfere with quantitation of drug levels in PK/TK analysis. Monoclonal antibody drug interference, especially for human IgG4 drugs, presents an additional challenge for ADA analysis due to its longer half-life and higher dose. Assay tolerance to such interference may depend on assay platform and reagents. Various approaches have been used to improve drug tolerance in ADA analysis but limited success was observed. We have developed a breakthrough novel method that uses Precipitation and Acid dissociation (PandA) to overcome drug interference in the ADA assay. The method principle is based on four components for detection of total ADA (free ADA and drug bound ADA) in the presence of drug in patient samples: (1) use excess drug to saturate free ADA to form drug bound ADA as drug:ADA complexes, (2) precipitate the complex using an agent such as PEG, (3) acid dissociate ADA from drug and immobilize (capture) free ADA (and free drug) under acidic conditions (without neutralization) onto a large capacity surface, and (4) detect free ADA (not the captured drug) using specific anti-human Ig detection reagent.In this manuscript, we are describing case studies for three humanized monoclonal antibodies (an IgG1 and two IgG4 drugs). The three drug specific PandA ADA assays resulted in complete recovery of ADA in samples containing drug levels in excess of those expected in patients, in contrast to the commonly used acid dissociation approach in ECL bridging assays. This breakthrough novel method shows significant improvement over the current approaches. In fact, the drug interference or under detecting of ADA in all three cases was eliminated. This assay principle could be used not only for ADA assays but also PK and biomarker (drug target) analysis in the presence of interference factors.

PTU-078 A prospective evaluation of adalimumab drug levels and anti-drug antibodies using two commercial elisa and the influence of 6-thioguanine nucleotides amongst patients with inflammatory bowel disease:

<h3>Introduction</h3> Some,^1,2but not all³studies have demonstrated a relationship between therapeutic drug monitoring (TDM) of adalimumab (ADA) and outcomes in inflammatory bowel disease (IBD). We evaluated the utility of TDM of ADA in patients with CD using two commercially available ELISA. <h3>Method</h3> ADA drug levels (DL) and anti-drug antibodies (ADAb) were measured in CD patients (n = 80) from 2 tertiary referral centres, between November 13 and February 14 using the Lisa-Tracker Duo ((LT) Theradiag, France) and Immundiagnostik ELISA ((IM) Germany). Faecal calprotectin (FC), C-reactive protein (CRP &lt;5 mg/L remission) and clinical activity (Harvey Bradshaw Index,(HBI) &lt;5 remission) were also recorded. LT kits were provided by Theradiag at no cost. <h3>Results</h3> Neither assay showed a significant difference in ADA DL between remission and active disease (See table). No significant differences in DL were observed in TDM performed at trough (day 13/14, n = 13) or at any other time in the treatment cycle, nor amongst those receiving ADA every other week compared to weekly. Thiopurine metabolites (TGN) were performed in 51/52 patients taking thiopurines,(median 302, IQR 242–411 pmol/8×10⁸). There was no significant difference between DL and TGN according to TGN quartiles. ADAb were detected in 1 (1.3%) patient using LT and 4 (5%) using IM. Concomitant immunomodulation or a therapeutic TGN (&gt;235) did not significantly influence median DL or the detection of ADAb with either assay. IM ADA showed proportional positive bias (79.6%) against LT kit (Passing Bablok regression IM = 1.74 LT–0.06). <h3>Conclusion</h3> No optimal cut-off could be identified that predicted clinical or biochemical remission or FC. Concomitant immunomodulation and TGN concentration was not associated with higher ADA DL. ADAb development was very rare whether measuring free (LT) or total (IM) ADAb. Further studies are needed to establish the cause of DL variation and understand differences in ADA pharmacokinetics in patients with CD. <h3>Disclosure of interest</h3> E. Johnston: None Declared, M. Ward: None Declared, B. Warner: None Declared, P. Irving Speaker Bureau of: AbbVie, MSD, Takeda, Warner Chilcott, Shire, Ferring and Tillotts Pharma. <h3>References</h3> Mazor Y, <i>et al</i>. Aliment Pharmacol Ther. 2014;40:620–628 Roblin X, <i>et al</i>. Clin Gastro Hepatol. 2014;12:80–84 Chiu Y, <i>et al</i>. Inflamm Bowel Dis. 2013;19:1112–1122

Immunogenicity of Anti-TNF-α Biotherapies: II. Clinical Relevance of Methods Used for Anti-Drug Antibody Detection.

Immunogenicity of biopharmaceuticals is complex and influenced by both structural and pharmacological factors, and by patient-related conditions such as disease being treated, previous and concomitant therapies, and individual immune responsiveness. Essential for tailored therapeutic strategies based on immunopharmacological evidence from individual patients (personalized medicine) is the use of assays for anti-drug antibodies (ADA) that are accurate and relevant in the clinical setting. This paper discusses immunogenicity of genetically engineered immunoglobulins directed against tumor-necrosis factor-α (TNF). Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies. The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists.

Tu1322 A Prospective Evaluation of Adalimumab Drug Levels and Anti-Drug Antibodies Using Two Commercial ELISA and the Influence of 6-Thioguanine Nucleotides Amongst Patients With Inflammatory Bowel Disease

Introduction Some, 1,2 but not all 3 studies have demonstrated a relationship between therapeutic drug monitoring (TDM) of adalimumab (ADA) and outcomes in inflammatory bowel disease (IBD). We evaluated the utility of TDM of ADA in patients with CD using two commercially available ELISA. Method ADA drug levels (DL) and anti-drug antibodies (ADAb) were measured in CD patients (n = 80) from 2 tertiary referral centres, between November 13 and February 14 using the Lisa-Tracker Duo ((LT) Theradiag, France) and Immundiagnostik ELISA ((IM) Germany). Faecal calprotectin (FC), C-reactive protein (CRP Results Neither assay showed a significant difference in ADA DL between remission and active disease (See table). No significant differences in DL were observed in TDM performed at trough (day 13/14, n = 13) or at any other time in the treatment cycle, nor amongst those receiving ADA every other week compared to weekly. Thiopurine metabolites (TGN) were performed in 51/52 patients taking thiopurines,(median 302, IQR 242–411 pmol/8×10 8 ). There was no significant difference between DL and TGN according to TGN quartiles. ADAb were detected in 1 (1.3%) patient using LT and 4 (5%) using IM. Concomitant immunomodulation or a therapeutic TGN (>235) did not significantly influence median DL or the detection of ADAb with either assay. IM ADA showed proportional positive bias (79.6%) against LT kit (Passing Bablok regression IM = 1.74 LT–0.06). Conclusion No optimal cut-off could be identified that predicted clinical or biochemical remission or FC. Concomitant immunomodulation and TGN concentration was not associated with higher ADA DL. ADAb development was very rare whether measuring free (LT) or total (IM) ADAb. Further studies are needed to establish the cause of DL variation and understand differences in ADA pharmacokinetics in patients with CD. Disclosure of interest E. Johnston: None Declared, M. Ward: None Declared, B. Warner: None Declared, P. Irving Speaker Bureau of: AbbVie, MSD, Takeda, Warner Chilcott, Shire, Ferring and Tillotts Pharma. References Mazor Y, et al . Aliment Pharmacol Ther. 2014;40:620–628 Roblin X, et al . Clin Gastro Hepatol. 2014;12:80–84 Chiu Y, et al . Inflamm Bowel Dis. 2013;19:1112–1122

Feasibility of immuno-PCR technology platforms as an ultrasensitive tool for the detection of anti-drug antibodies.

During the early clinical development of a receptor-IgG1 fusion protein (Drug X), a risk based strategy was utilized to evaluate immunogenicity from Phase I through Proof of Concept clinical studies. Three different technology platforms, enzyme-linked immunosorbant assay, electrochemiluminescent assay and newly emerging immuno-PCR were utilized for evaluation of immunogenic response. An ELISA with adequate sensitivity but significant drug interference was replaced by electrochemiluminescent method with improved drug tolerance; however, an inverse correlation was observed between the administered dose and the incidence of anti-drug antibodies. Further evaluation of an immuno-PCR showed reduced drug interference enabling the detection of anti-drug antibodies in the presence of excess amount of Drug X. Immuno PCR assay proved to be a feasible platform for anti-drug antibody characterization.

How Useful is Anti-TNF Serum Level and Anti-drug Antibodies Detection in Evaluating Patients with Spondyloarthritis?

Objective: The aim of this study was to assess whether infliximab and adalimumab drug serum levels and the detection of anti-drug antibodies can be of use in better observing disease activity in patients with spondyloarthritis, besides classical tools such as BASDAI, ASDAS and inflammatory markers. We proposed to evaluate the influence of ADA in non-responders and in drug-related adverse events. Methods: Over one year, we enrolled 115 patients with SpA, treated with infliximab or adalimumab. Patients who delayed prescribed drug administration were excluded from the study cohort. The population comprised 69 patients - 33 on IFX and 35 on ADA. NSAIDs administration was recommended “on demand”. Demographic, clinical (BASDAI, ASDAS) and laboratory (ESR, CRP) data was collected together with drug serum level and anti-drug antibodies using ELISA. The statistical analysis was performed using the SPSS software, version 20.0 with the aid of Student t-test, Spearman and Pearson tests. Results: Detectable IFX serum levels were identified in 60% of patients while 40% had undetectable drug titers. The IFX-negative had significantly higher disease activity scores: BASDAI (P=0.023), ASDAS-ESR (P<0.001) and ASDAS-CRP (P<0.001). Significant differences were found in the same subgroups regarding inflammatory markers, with higher ESR (P<0.001) and CRP (P=0.032) in patients with undetectable IFX levels. When measuring ADL serum levels, 82% had detectable drug concentrations, with lower BASDAI (P<0.001), ASDAS-ESR and ASDASCRP (P<0.001) and higher ESR and CRP at collection time when compared to ADL-negative patients. NSAID consumption correlated to undetectable levels of IFX and ADL as well as with anti-drug antibodies for both IFX and ADL positivity. All patients who experienced drug related adverse events on both IFX and ADL had positive anti-drug antibodies. Conclusion: Serum drug level measurement and anti-drug antibody detection can be used as a completion of a clinician’s tools in assessing disease activity, leading to an optimal patient management.